The antigen-antibody interaction is a bimolecular association similar to an enzyme-substrate interaction, with the important distinction that it is a reversible process. The interactions between an antibody and an antigen are governed by various noncovalent interactions between the antigenic determinant, or epitope, of the antigen and the variable-region domain of the antibody molecule. The specificity of an antibody for an antigen has led to the development of a variety of immunologic assays which can be used to detect the presence of antibody or antigen. These assays have been instrumental in diagnosing diseases, monitoring the level of the humoral immune response, and identifying molecules of biological interest.
Antigens are routinely detected on membranes (Western blots) and in situ (immunohistochemistry, immunofluorescence, immunostaining, etc.) There are many variations on the available methods of detecting antigens, depending on the number and types of antibodies used, the label and the substrate. Independent of the variation, antigen detection essentially depends upon a specific antibody-antigen reaction forming an antibody-antigen complex.
The noncovalent interactions that comprise antigen-antibody binding include hydrogen bonds, and ionic, hydrophobic and van der Waals interactions, each of which is relatively weak in comparison to a covalent bond, and with each effective interaction operating over a very small distance. Therefore, a strong antigen-antibody interaction requires a large number of such associations, and a very tight fit between the antigen and antibody, owing to the high degree of specificity which is characteristic of antigen-antibody interactions.
The detection of the primary antibody-antigen complex has been demonstrated in numerous ways. Detection methods include directly labeled monoclonal antibody, wherein the label consists of an enzyme, e.g., alkaline phosphatase (AP), and Horseradish Peroxidase (HRP); a fluorochrome (a fluorescent compound), e.g., fluorescein, rhodamine, Texas Red, Cy-3, and Cy-5; a heavy metal chelate such as europium, lanthanum, yttrium, and gold; a radioactive isotope such as .sup.125 I, .sup.131 I, .sup.3 H, .sup.14 C, and .sup.35 S; or the label may be a secondary reporter, e.g., biotin, streptavidin, avidin, digoxigenin, or dinitrophenyl. Alternatively, detection methods may also include directly labeled polyclonal antibody, wherein the label may consist of the above-identified elements listed for monoclonal antibodies. Further, labeled secondary antibody which is polyclonal anti-first antibody, such as goat anti-mouse IgG-conjugate, may be used as a method of detection. Other detection methods include the use of labeled secondary reagent which is not necessarily an antibody, such as AP-streptavidin; labeled secondary antibody which is anti-conjugated epitope, such as HRP-goat-antifluorescein and AP-rabbit-anti-DNP; and unlabeled secondary antibody, detected with a labeled tertiary antibody or labeled tertiary component.
In extracts where the antigenic proteins represent only a tiny fraction of the total protein, the number and sizes of proteins with a particular epitope can be rapidly determined by Western blotting. Western blotting consists of electrophoretic transfer of an antigenic protein or proteins from a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) onto a nitrocellulose filter placed on one face of the gel, and as the protein is transferred, its position on the SDS-PAGE gel is preserved. The transferred protein binds tightly and non-covalently to the nitrocellulose, and can be exposed to a primary antibody that will bind to it. This bound primary antibody can then be bound by a secondary antibody containing a visualizable, covalently attached marker. If labeled specific antibody is not available, antigen-antibody complexes can be detected by adding a secondary anti-isotope antibody that is either radiolabeled or enzyme-labeled, and the band is visualized by autoradiography or substrate addition. Only those proteins with the epitope will be visualized in this manner, and if several proteins with different molecular weights have the epitope, each will be seen as a separate band on the nitrocellulose (S. Hockfield, et al., Selected Methods for Antibody and Nucleic Acid Probes, Cold Spring Harbor Laboratory Press, 1993, pp. 293-316).
Western blotting can identify either a given protein antigen or specific antibody. For example, Western blotting has been used to identify the envelope and core proteins of HIV and the antibodies to these components in the serum of HIV-infected individuals.
Immuno-PCR, a hybrid of PCR and immunoassay systems, combines the versatile molecular recognition of antibodies with the amplification potential of DNA replication. The technique involves the in situ assembly of the labeled DNA-antibody complex during the assay, creating variable stoichiometry in both the attachment of the DNA label, and the assembly of the components.
The procedural complexity of immuno-PCR has been reduced by the direct chemical attachment of DNA to analyte antibodies, whereby immobilized capture antibodies and a reporter antibody that carries a covalently attached DNA label are used, and the assay response is obtained by PCR of the DNA label and detection of the amplification products. This technique has been modified to develop an immuno-PCR sandwich assay for multiple analytes (see R. D. Joerger, et al., Clinical Chemistry, 1995, 41 (9): 1371-1377; E. R. Hendrickson, et al., Nucl. Acids Res., 1995, 23 (3): 522-529; and T. Sano, et al., Science, 1992, 258: 120-122).
However, immuno-PCR, albeit exhibiting enhanced sensitivity over traditional methods, is time consuming, complex and it does not lend itself to automation.
In order to detect a specific nucleic acid sequence, a highly specific probe DNA or RNA sequence (which is complementary to all or part of the sequence to be determined) is isolated, amplified by cloning, purified to homogeneity and labeled with a suitable marker. The purified, labeled DNA is added to a hybridization solution containing denatured nucleic acids (RNA or DNA) from a sample to be tested. The aqueous conditions of the hybridization solution are adjusted to allow nucleic acid hybridization or reannealing, thereby allowing the labeled molecules to hybridize with unlabeled, complementary sequence counterparts. Duplex formation can be monitored by digestion with single strand-specific nucleases (such as S1 nuclease). Recovery and quantitation of resistant, i.e., double-stranded, reannealed material provides a measure of the nucleic acid sequence tested for. The amount of hybridization is a function of the initial concentration of DNA and the time allowed for reannealing. Therefore, increased initial DNA concentrations can lead to substantially reduced hybridization times.
An example of the use of specific hybridization to detect sequences of oligonucleotides is that described by Southern, E., J. Mol. Biol. 98: 503, 1975. In this assay, a sample containing the DNA sequence to be detected is purified, digested with appropriate restriction endonucleases, and the fragments separated by gel electrophoresis. The fragments are then bound to a suitable solid support, such as nitrocellulose. This binding takes a minimum of 12 to 16 hours in the presence of a solution containing a relatively high concentration of sodium chloride. A labeled probe, complementary to the sequences to be determined, is then added to the nitrocellulose and allowed to hybridize for a period of 12 to 48 hours. After this period of time, the nitrocellulose must be washed under appropriate salt and temperature conditions, since otherwise the labeled probe will bind nonspecifically to both the membrane and to other non-homologous DNA sequences, leading to background "noise" or "false positives".
In a simplified version of the above-identified Southern hybridization assay, nucleic acid samples to be analyzed are "dotted" onto a solid support in an unfractionated state. The solid support is then probed as in the Southern hybridization assay, washed, and the amount of bound probe is quantitated.
However, there is no teaching in the prior art of using the above-identified techniques as a means to monitor the presence of an antigen of interest in a Western blot assay, without the additional requirement of PCR of the DNA label and detection of the amplification products.
U.S. Pat. Nos. 5,175,270 and 5,487,973 to Nilsen et al., herein incorporated by reference, are directed to dendrimers, a class of reagents for assaying nucleic acid sequences which are comprised of successive layers of polynucleotides, including a double-stranded waist and single-stranded, free arms at the molecule's ends, formed by hybridization of the arms to adjacent molecule arms. The outer layer polynucleotides are specific for the sequence to be identified, through their non-annealed, free, single-stranded arms.
It would be advantageous to develop a method of antibody detection which employs a Western blot technique with the oligonucleotide-antibody conjugates and the sensitivity of DNA dendrimers, without the necessity of performing PCR of the oligonucleotide label in order to detect the antigen-antibody complex.